Patent application title: METHOD FOR ENHANCING EFFICACY AND SELECTIVITY OF CANCER CELL KILLING BY DNA DAMAGING AGENTS
Inventors:
Derek J. Hoelz (Bangor, ME, US)
IPC8 Class: AA61K31713FI
USPC Class:
514 34
Class name: O-glycoside oxygen of the saccharide radical bonded directly to a polycyclo ring system of three or more carbocyclic rings oxygen of the saccharide radical bonded directly to a polycyclo ring system of four carbocyclic rings (e.g., daunomycin, etc.)
Publication date: 2015-03-05
Patent application number: 20150065444
Abstract:
The invention relates to the treatment of cancer using DNA damaging
agents. The invention provides methods for treating a mammal with cancer,
the method comprising inhibiting in the mammal acidic residue
methyltransferase (Arm1) in combination with administering to the mammal
a DNA damaging agent. The invention further provides pharmaceutical
formulations comprising an inhibitor of acidic residue methyltransferase
(Arm1) and a DNA damaging agent.Claims:
1. A method for treating a mammal with cancer, the method comprising
administering to the mammal a pharmaceutical formulation comprising an
inhibitor of acidic residue methyltransferase (Arm1) in combination with
a DNA damaging agent, wherein the inhibitor of Arm1 enhances the efficacy
and selectivity of cancer cell killing by the DNA damaging agent.
2. (canceled)
3. A method for enhancing efficacy and selectivity of cancer cell killing by a DNA damaging agent, comprising contacting a tumor cell with an inhibitor of Arm1 in combination with a DNA damaging agent, wherein the inhibitor of Arm1 enhances the efficacy and selectivity of cancer cell killing by the DNA damaging agent.
4. The method of claim 3, wherein the cancer cell is in the body of a mammal.
5. The method of claim 3, wherein the DNA damaging agent is selected from the group consisting of doxorubicin, 6-mercaptopurine, gemcitabine, cyclophosphamide, melphalan, busulfan, chlorambucil, mitomycin, cisplatin, bleomycin, dectinomycin, irinotecan, and mitoxantrane.
6. The method of claim 1, wherein the DNA damaging agent is selected from the group consisting of doxorubicin, 6-mercaptopurine, gemcitabine, cyclophosphamide, melphalan, busulfan, chlorambucil, mitomycin, cisplatin, bleomycin, dectinomycin, irinotecan, and mitoxantrane.
Description:
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to the use of DNA damaging agents for the treatment of cancer.
[0004] 2. Summary of the Related Art
[0005] DNA damaging agents, such as doxorubicin, have been widely used in the treatment of cancer. Such agents selectively kill proliferating cells while being less toxic to non-proliferating cells, thus providing some measure of cancer cell selectivity, since most cells of the body are non-proliferating. However, important normal cell types, such as intestinal endothelium, immune system cells, bone marrow cells and hair follicle cells do proliferate, and thus are also killed by DNA damaging agents, leading to numerous unwanted side effects. There is, therefore a need to improve the efficacy and selectivity of DNA damaging agents for the treatment of cancer.
BRIEF SUMMARY OF THE INVENTION
[0006] The invention relates to the treatment of cancer using DNA damaging agents. The inventor has surprisingly discovered that knockdown of a previously uncharacterized gene, acidic residue methyltransferase (Arm1), improves the ability of cells having a wild-type p53 gene to survive treatment with DNA damaging agents, while causing cells having mutant p53 genes to become more sensitive to killing by DNA damaging agents. Since more than 50% of cancer cell types have mutant p53 genes, while normal proliferating cells have wild type p53 genes, inhibition of Arm1 increases both the efficacy and selectivity of DNA damaging agents for killing cancer cells.
[0007] In a first aspect, the invention provides a method for treating a mammal with cancer, the method comprising inhibiting in the mammal acidic residue methyltransferase (Arm1) in combination with administering to the mammal a DNA damaging agent.
[0008] In a second aspect, the invention provides a pharmaceutical formulation comprising an inhibitor of acidic residue methyltransferase (Arm1) and a DNA damaging agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1a shows that a carboxyl methyltransferase targets PCNA in MDA MB 468 cells. FIG. 1b shows that SAM-dependent methyltransferase domains exist in the C6orf211 protein (SEQ ID NOS: 1-8, respectively). FIG. 1c shows the positions of the motifs I, II and regions II and III in CheR and the C6orf211 protein. FIGS. 1d and 1e show an illustrative representation of the SAM-MT fold in the C-terminus of the CheR. Figure if shows recombinant 6× His-tagged Arm1 isolated from Tni insect cell extracts and analyzed by SDS-PAGE and colloidal Coomassie Blue staining FIG. 1g shows that Arm possesses a PCNA-dependent carboxyl methyltransferase activity.
[0010] FIG. 2a shows that Doxorubicin (Dox) promotes PCNA methyl esterification in MCF7 cells. FIG. 2b shows that PCNA-dependent methyltransferase activity is altered following Dox treatment. FIG. 2c shows that Dox induces p21 expression in MCF7 cells. FIG. 2d shows that p21 binding promotes PCNA methyl esterification. FIG. 2e shows that the p21-induced basic shift is a result of PCNA methyl esterification. FIG. 2f shows that p21 does not interact with Arm1. FIG. 2g shows that PCNA does not directly interact with Arm1 in SK-Br-3 cells.
[0011] FIG. 3a shows that PCNA-dependent methyltransferase activities are altered in shRNA expressing SK-Br-3 and MCF7 cells. FIG. 3b shows that DNA damage sensitivity in Arm1 knockdown cells is related to p53 status. FIG. 3c shows that Arm1 knockdown promotes DNA repair.
[0012] FIG. 4a shows that PCNA chromatin stability is unaffected by Arm1. FIG. 4b shows that Arm1 is recruited to the chromatin and promotes PCNA ubiquitylation. FIG. 4c shows that Arm1-dependent methyltransferase activity promotes Rad18's interactions with Arm1 and PCNA. FIG. 4d shows that Arm1 interacts with Rev1.
[0013] FIGS. 5a and b show a model for PCNA methyl esterification.
[0014] FIG. 6a shows fractions from passage over a Superdex S200 gel filtration column. FIG. 6b shows active fractions resolved by 2D-PAGE and stained with colloidal Coomassie Blue. The position of the 50 kDa product of the uncharacterized gene C6orf211 is identified with an arrow. FIG. 6c shows proteins present in the enriched fractions identified by mass spectrometry and grouped by cellular function.
[0015] FIG. 7 (SEQ ID NOS: 9-11, respectively) shows sequence alignments of the CheR, C6orf211, and PIMT proteins. Amino acids are colour coded green (polar), red (nonpolar, hydrophobic), pink (basic), and blue (acidic).
[0016] FIG. 8 (SEQ ID NOS: 12-19, respectively) shows alignment of the C6orf211 proteins from eight eukaryotic organisms with motifs I and II and regions II and III identified.
[0017] FIG. 9 (SEQ ID NOS: 20-25, respectively) shows patterns of methyl esterification of peptides from p21-PIP affinity purified PCNA isoforms separated and excised from 2D-PAGE gels and analyzed by LC-MS/MS. Positively identified peptide sequences are shown in black and unobserved sequences are shown in red. The locations of methyl esterified residues in the PCNA isoform spots are presented in bolded blue.
[0018] FIG. 10a shows shRNA expression. FIG. 10b shows reduction of Arm1 mRNA expression in shRNA expressing cells.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0019] The invention relates to the treatment of cancer using DNA damaging agents. The inventor has surprisingly discovered that knockdown of a previously uncharacterized gene, acidic residue methyltransferase (Arm1), improves the ability of cells having a wild-type p53 gene to survive treatment with DNA damaging agents, while causing cells having mutant p53 genes to become more sensitive to killing by DNA damaging agents. Since more than 50% of cancer cell types have mutant p53 genes, while normal proliferating cells have wild type p53 genes, inhibition of Arm1 increases both the efficacy and selectivity of DNA damaging agents for killing cancer cells.
[0020] In a first aspect, the invention provides a method for treating a mammal with cancer, the method comprising inhibiting in the mammal acidic residue methyltransferase (Arm1) in combination with administering to the mammal a DNA damaging agent.
[0021] "Treating a mammal with cancer" means causing in the mammal a reduction of signs or symptoms of cancer.
[0022] "Inhibiting acidic residue methyltransferase 1 (Arm1)" means reducing the activity and/or expression of Arm1. Preferred methods of inhibiting Arm1 include, without limitation, contacting a cancer cell with a small molecule inhibitor of Arm1 activity, or a dominant negative mutant of Arm1, such as an Arm1 protein with some but not all of its protein- or substrate-interactive domains inactivated or a genetic suppressor element (GSE) that encodes a fragment of the Arm1 protein, which interferes with the Arm1 activity. Contacting a tumor cell with a dominant negative mutant of Arm1 includes expressing the dominant negative mutant via transfection with a virus or a vector expressing the dominant negative mutant, or contacting a cancer cell with a peptide encoded by the GSE. Additional preferred methods include contacting a cell with an inhibitor of Arm1 gene expression, including without limitation, a short hairpin RNA (shRNA), a small inhibitory RNA (siRNA), an antisense nucleic acid (AS) and a ribozyme. "Contacting a tumor cell with an inhibitor of Arm1 gene expression" includes exogenously providing to a cell an inhibitor of Arm1 gene expression, as well as expressing an inhibitor of Arm1 gene expression in a cell. Expressing an inhibitor of gene expression in a cell is conveniently provided by transfection with a virus or a vector expressing such an inhibitor.
[0023] "Administering to the mammal a DNA damaging agent" means providing the mammal with a DNA damaging agent by any medically acceptable route, including, without limitation, parenteral, oral, sublingual, transdermal, topical, intranasal, intratracheal, or intrarectal. In certain preferred embodiments, compositions of the invention are administered parenterally, e.g., intravenously in a hospital setting. In certain other preferred embodiments, administration may preferably be by the oral route. Preferred DNA damaging agents include, without limitation, doxorubicin, 6-mercaptopurine, Gemcitabine, Cyclophosphamide, Melphalan, Busulfan, Chlorambucil, Mitomycin, Cisplatin, Bleomycin, Dectinomycin, Irinotecan and Mitoxantrane.
[0024] In combination with means in the course of treating the same disease in the same mammal, and includes inhibiting Arm1 and administering the DNA damaging agent in any order, including simultaneous administration, as well as any temporally spaced order, for example, from sequentially with one immediately following the other to up to several hours apart. The administration of an inhibitor of Arm1 and DNA damaging agent may be by the same or different routes.
[0025] In the methods for treatment according to the invention, the compounds and other inhibitors described above may be incorporated into a pharmaceutical formulation. Such formulations comprise the compound, which may be in the form of a free acid, salt or prodrug, in a pharmaceutically acceptable diluent, carrier, or excipient. Such formulations are well known in the art and are described, e.g., in Remington's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, Pa., 1990.
[0026] The characteristics of the carrier will depend on the route of administration. As used herein, the term "pharmaceutically acceptable" means a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism, and that does not interfere with the effectiveness of the biological activity of the active ingredient(s). Thus, compositions according to the invention may contain, in addition to the inhibitor, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
[0027] As used herein, the term "pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the above-identified compounds and exhibit minimal or no undesired toxicological effects. Examples of such salts include, but are not limited to, salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, polygalacturonic acid, and the like. The compounds can also be administered as pharmaceutically acceptable quaternary salts known by those skilled in the art, which specifically include the quaternary ammonium salt of the formula --NR+Z--, wherein R is hydrogen, alkyl, or benzyl, and Z is a counterion, including chloride, bromide, iodide, --O-alkyl, toluenesulfonate, methylsulfonate, sulfonate, phosphate, or carboxylate (such as benzoate, succinate, acetate, glycolate, maleate, malate, citrate, tartrate, ascorbate, benzoate, cinnamoate, mandeloate, benzyloate, and diphenylacetate).
[0028] The active compound is included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount without causing serious toxic effects in the patient treated. The effective dosage range of the pharmaceutically acceptable derivatives can be calculated based on the weight of the parent compound to be delivered. If the derivative exhibits activity in itself, the effective dosage can be estimated as above using the weight of the derivative, or by other means known to those skilled in the art.
[0029] In a second aspect, the invention provides a pharmaceutical formulation comprising an inhibitor of acidic residue methyltransferase (Arm1) and a DNA damaging agent. Preferred inhibitors of Arm1 include, without limitation, small molecule inhibitors of Arm1 activity, dominant negative mutants of Arm1, such as an Arm1 protein with some but not all of its protein- or substrate-interactive domains inactivated, genetic suppressor elements (GSEs) that encodes a fragment of the Arm1 protein, which interferes with the Arm1 activity.
[0030] Preferred DNA damaging agents include, without limitation, doxorubicin, 6-mercaptopurine, Gemcitabine, Cyclophosphamide, Melphalan, Busulfan, Chlorambucil, Mitomycin, Cisplatin, Bleomycin, Dectinomycin, Irinotecan and Mitoxantrane.
[0031] The pharmaceutical formulation may further comprise additional diluents, excipients or carriers, as described above for the first aspect of the invention.
[0032] The following examples are intended to further illustrate certain preferred embodiments of the invention and are not to be construed as limiting the scope of the invention.
Example 1
Cell Culture
[0033] MCF7 and SK-Br-3 cells were obtained from ATCC and maintained in DMEM or McCoys 5A supplemented with 10% FBS and antibiotics at 37° C., 5% CO2. A human Flag-tagged Arm1, Rev1, and p21 expression construct (Origene) were transiently transfected into SK-Br-3 cells with Fugene 6 (Roche) and extracts generated after 24 h. Lentiviral shRNA particles were obtained from Open Biosystems and stably expressing clones selected with puromycin and confirmed by GFP expression and Q-PCR (FIG. 10). Clonogenic survival assays were performed following exposure to Dox (Sigma) or UV-C using a Spectrolinker (Spectronics) for 4 h. Surviving colonies were stained with methylene blue and counted 2 weeks after treatments.
Example 2
Vapour Diffusion Assay
[0034] The assay was performed as previously described.37 Cell extracts were assayed with [3H-methyl]-SAM (NEN) for 1 h before equilibration with 100 mM NaOH with 1% SDS and spotting onto filter paper folded into an accordion pleat and placed above scintillation fluid. Diffused 3H-methanol was detected the following day.
Example 3
Protein Expression and Purification
[0035] Chromatography was performed using a Biologic DuoFlow (BioRad) using phenyl Sepharose HP (HiTrap) and Superdex S200 columns (GE Biosciences). Recombinant PCNA was expressed either as a calmodulin binding peptide fusion (CBPPCNA) using the pDual expression system and purified using calmodulin agarose (Stratagene) or a 6× His-tagged fusion expressed in pET303/CT-His (InVitrogen) and purified with Ni2+ Sepharose (GE Biosciences). His-tagged human Arm1 was cloned into a baculovirus expression vector and expressed in Tni insect cells (Allele Biotech., Inc.). GST, GST-p21, GST-p21(PIP), and GST-Rad18 were expressed in BL21(DE3) cells and isolated using glutathione Sepharose (GE Biosciences). GST-p21 was isolated from inclusion bodies as described38. Anti-Flag immunoprecipitations were performed with anti-Flag M2 Affinity Gel (Sigma). p21(PIP)-affinity beads were generated by covalently coupling a synthetic peptide (Anaspec) to CH-Sepharose (GE Biosciences).
Example 4
Electrophoresis and Mass Spectrometry
[0036] 2D-PAGE and protein identification and sequencing by LC-MS/MS was performed as previously described10. Anti-PCNA (PC10) were from Millipore, anti-histone H3 were from Cell Signalling, anti-p21 (C19) and anti-α-tubulin antibodies were from Santa Cruz Biotech, anti-DDK (Flag) antibodies were from Origene, anti-C6orf211 antibodies were from Sigma, and anti-Rad18 was from ThermoElectron.
Example 5
C6orf211 Encodes a PCNA-Dependent Carboxyl Methyltransferase (Arm1)
[0037] The results from this example are shown in FIG. 1. a, A carboxyl methyltransferase targets PCNA in MDA MB 468 cells. SAM-dependent carboxyl methyltransferase activities of whole cell extracts (WCE) were detected by vapour diffusion assay37. Average activities are presented (±S.E.M). D heat denatured (95° C., 5 min); CBP-PCNA, calmodulin binding peptide (CBP)-tagged PCNA; bovine serum albumin (BSA). Significance was determined using an unpaired two-tailed t-test. b, SAM-dependent methyltransferase domains exist in the C6orf211 protein. Bacterial CheR methyltransferase sequences containing motifs I and II and region III were aligned to the full-length C6orf211 protein sequence using KALIGN39 and align sequences shown. Conserved glycine and glutamic acid residues in CheR motif I and catalytic aspartic acid and conserved isoleucine residues of motif II are underlined. Full-length CheR and C6orf211 protein sequences were aligned with Nomad40. CheR's region II sequence is underlined. c, The positions of motifs I, II and regions II and III in CheR and the C6orf211 protein. d, Illustrative representation of the SAM-MT fold in the C-terminus of the CheR13. e, The C-terminus of the C6orf211 protein (a.a. 227-441) has the potential for a SAM-MT fold. Secondary structures predicted with Jpred41 were assembled into a hypothetical SAM-MT fold. Conserved a-helices (A-E) are shown in yellow and b-sheets (1-7) are in magenta. The CheR structure lacks a-helix C. The positions of motifs I and II in CheR and Arm1 structures are highlighted in red and blue, respectively. f, Recombinant 6× His-tagged Arm1 was isolated from Tni insect cell extracts and analyzed by SDS-PAGE and colloidal Coomassie Blue staining. g, Arm possesses a PCNA-dependent carboxyl methyltransferase activity. Purified Arm1 was assayed in the absence and presence of purified 6× His-tagged PCNA using the vapour diffusion assay. Background (His-PCNA alone) subtracted counts from three independent experiments are shown (±S.E.M). Significance was determined using a two-tailed t-test.
Example 6
PCNA Methyl Esterification is Promoted by DNA Damage
[0038] The results from this example are shown in FIG. 2. a, Doxorubicin (Dox) promotes PCNA methyl esterification in MCF7 cells. Whole cell extracts (WCE) from cultures treated with Dox (5 mM) were resolved by 2D-PAGE followed by PCNA immunoblotting (IB). The basic-shifted PCNA isoform (ME-PCNA) is identified with an arrow. b, PCNA-dependent methyltransferase activity is altered following Dox treatment. Dox treated MCF7 WCE were assayed for PCNA-dependent activity in triplicate using the vapour diffusion assay, and average activities are presented ±S.E.M. Significance was determined using a two-tailed t-test. c, Dox induces p21 expression in MCF7 cells. Extracts were separated by SDS-PAGE and immunoblotted for p21.sup.Waf1/Cip1. d, p21 binding promotes PCNA methyl esterification. Untreated MCF7 extracts were incubated with GST, GST-p21 (full-length), and GST-p21(PIP) proteins bound to glutathione Sepharose for 2 h at 4° C. prior to 2D-PAGE and PCNA immunoblotting. e The p21-induced basic shift is a result of PCNA methyl esterification. p21(PIP) peptide was covalently coupled to CH-Sepharose and used to affinity purify PCNA from MCF7 whole cell extracts. Purified fractions were separated by 2D-PAGE and stained with colloidal Coomassie blue. Spots were excised from the gel, digested with trypsin, and analyzed by LC-MS/MS. Protein spots identified as PCNA (A-F) were further scrutinized for presence of methyl esters (table I). Methyl esterification of 16 highly conserved glutamate residues and one aspartate residue was observed (FIG. 10). f, p21 does not interact with Arm1. SK-Br-3 cell extracts expressing Flag (vector control) or Flag-p21 were immunoprecipitated with anti-Flag antibodies. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-Flag and anti-Arm1 antibodies. g, PCNA does not directly interact with Arm1 in SK-Br-3 cells. SK-Br-3 cell extracts expressing Flag (vector control) or Flag-Arm1 were immunoprecipitated with anti-Flag antibodies. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted for Flag and PCNA.
TABLE-US-00001 TABLE I Enrichment of Methyltransferase Activity TOTAL VOLUME TOTAL UNITS PROTEIN SPECIFIC ACTIVITY PROTEIN FRACTION (ML) (FMOL/MIN) (MG) (FMOL/MG/MIN) Lysatea 4 58.3 20 2.9 30% NH4SO4a 4 48 12 4.0 Phenyl Sepharose HPab 5 40 1.7 23.5 Superdex S200b 1 39 0.38 102.6 aMethyl esterification of endogenous proteins was subtracted. bActivities of pooled peak fractions.
Example 7
Arm1-Dependent PCNA Methyl Esterification is Linked to DNA Repair
[0039] The results from this example are shown in FIG. 3. a, Arm1 promotes PCNA methyl esterification following DNA damage. PCNA was analyzed by 2D-PAGE immunoblotting in SK-Br-3 and MCF7 cells expressing Arm1 or non-targeting (control) shRNA in the absence of DNA damage or 4 h following Dox treatment or UV irradiation. b, PCNA-dependent methyltransferase activities are alter in shRNA expressing SK-Br-3 and MCF7 cells. WCE were assayed for PCNA-dependent carboxyl methyltransferase activity in the presence of His-PCNA (2 mg) and average activities shown (±S.E.M.). Significance was determined using a two-tailed t-test. c, DNA damage sensitivity in Arm1 knockdown cells is related to p53 status. MCF7 (p53 wild-type) and SK-Br-3 (p53-mutant) cells were exposed to increasing levels of Dox and UV and survival determined by clonogenic survival. Average results from three independent experiments are presented ±SD. d, Arm1 knockdown promotes DNA repair. DNA repair rates were determined in shRNA expressing SK-Br-3 and MCF7 cells using host cell reactivation assay. Cells were transfected with UV-irradiated reporter plasmids and average repair rates were determined after 24 h. Average results from independent experiments are presented (±S.E.M.). Significance was determined with a two-tailed t-test.
Example 8
Arm1 Promotes DNA Damage Tolerance
[0040] The results from this example are shown in FIG. 4. a, PCNA chromatin stability is un affected by Arm1. MCF7 and SK-Br-3 cell expressing shRNA were UV-irradiated (20 J/m2) for the indicated times and fractionated to Triton X-100 soluble and chromatin bound insoluble fractions. Fractions were separated by SDS-PAGE and immunoblotted for PCNA, Arm1, and p21. a-tubulin and histone H3 were used as loading controls. b, Arm1 is recruited to the chromatin and promotes PCNA ubiquitylation. MCF7 cells expressing Arm1 shRNA were UV-irradiated (20 J/m2) and proteins cross-linked with DTBP at the indicated times prior to Triton X-100 extraction. Proteins present in the insoluble fraction were analyzed by SDS-PAGE and immunoblotting for PCNA and Arm1. Histone H3 served as a loading control. c, Arm1-dependent methyltransferase activity promotes Rad18's interactions with Arm1 and PCNA. Purified recombinant PCNA was incubated in the absence and presence of purified recombinant Arm1 with and without SAM (10 mM) or sinefungin (20 mM) for 1 h at 37° C. prior to rocking with glutathione Sepharose bound GST-Rad18 for 15 m at 4° C. The GST-Rad18 beads were washed and analyzed by SDS-PAGE and immunoblotting. d, Arm1 interacts with Rev1. SK-Br-3 cells were transfected with control or Flag-Rev1 expression plasmids and incubated for 24 h. Cells were harvested 6 h after UV irradiation.
Example 9
Model for PCNA Methyl Esterification
[0041] The results from this example are shown in FIG. 5. Methyl esterified PCNA residues identified by LC-MS/MS sequencing of the p21-PIP affinity purified isoforms (FIG. 3d, table I, and FIG. 10) are shown (orange) on the PCNA structure described in Gulbis et al.21. PCNA subunits are shown in blue, green and magenta, and the p21 PIP-box peptide is shown in red. DNA damage induces up-regulation of p21 and methyl esterification of PCNA. Knock-down of Arm1 expression promotes survival in p53 wild-type cells and cytotoxicity in p53-mutant cells.
Example 10
Identification of the C6orf211 Protein in the PCNA-Dependent Carboxyl Methyltransferase Active Fraction
[0042] The results from this example are shown in FIG. 6. MDA MB468 cell extracts were subjected to 30% NH4SO4 precipitation prior to loading onto a phenyl Sepharose column elution with a linear gradient of NH4SO4 to 0% (a). Activity was further enriched by passage over a Superdex 5200 gel filtration column, and the active fractions resolved by 2D-PAGE and stained with colloidal Coomassie Blue (b). The position of the 50 kDa product of the uncharacterized gene C6orf211 is identified with an arrow. Proteins present in the enriched fractions were identified by mass spectrometry and grouped by cellular function (c).
Example 11
Sequence Alignments of CheR, C6orf211, and PIMT Proteins
[0043] The results from this example are shown in FIG. 7. Full-length protein sequences of CheR, the C6orf211 gene product, and PIMT were aligned using MUSCLE42. Consensus sequences previously determined in CheR and PIMT are shown13,14. Amino acids are colour coded green (polar), red (nonpolar, hydrophobic), pink (basic), and blue (acidic).
Example 12
Conservation of the C6orf211 Gene Product in Eukaryotes
[0044] The results from this example are shown in FIG. 8. The C6orf211 proteins from eight eukaryotic organisms were aligned with KALIGN and motifs I and II and regions II and III identified. The motif I sequence shows high conservation among all organisms and its location in the primary sequence positions it in the b1/aA loop of the hypothetical SAM-MT fold shown in FIG. 1e. Conserved glycine residues of motif I are underlined. Identification of motif II was made using the hypothetical SAM-MT fold. The motif II sequence present in the b2/aB loop is also highly conserved among all species. Regions II and III, although less conserved, show significant conservation.
Example 13
Methyl Esterification of p21-PIP Affinity Purified PCNA Isoforms
[0045] The results from this example are shown in FIG. 9. Affinity purified PCNA isoforms (FIG. 2) were separated and excised from 2D-PAGE gels and analyzed by LC-MS/MS. Positively identified peptide sequences are shown in black and unobserved sequences are shown in red. The locations of methyl esterified residues in the PCNA isoform spots are presented in bolded blue.
Example 14
Lentiviral shRNA Knock-Down of Arm Expression in MCF7 and SK-Br-3 Cells
[0046] The results from this example are shown in FIG. 10. a, MCF7 and SK-Br-3 cells were infected with lentiviral shRNA and bicistronic expression of TurboGFP confirmed shRNA expression. b, Arm1 mRNA expression is reduced in shRNA expressing cells. Relative Arm1 mRNA expression was determined by Q-PCR. Normalized expression levels are presented (±SD).
Example 15
Identification of Arm1
[0047] To determine if PCNA methyl esters were the result of a posttranslational mechanism we examined breast cancer cell extracts for it ability to methyl esterify PCNA (FIG. 1a). As a result we were able to detect a carboxyl methyltransferase activity in these extracts that was dependent on PCNA. To identify the enzyme responsible for methyl esterifying PCNA we subsequently enriched for PCNA-dependent carboxyl methyltransferase activity from extracts (FIG. 6 and table I). Using proteomics techniques then identified proteins comprising the active fractions, and the majority of proteins identified were of known function and were excluded from further consideration. From this approach we were able to rapidly narrow down methyltransferase candidates to an uncharacterized protein, the 50 kDa product of a hypothetical orf on chromosome 6 (C6orf211)12. This hypothetical protein was further assessed for methyltransferase potential.
[0048] As an initial step we aligned the C6orf211 protein with the bacterial glutamyl methyltransferase CheR and the human isoaspartate methyltransferase PIMT (FIG. 7). Like many of the SAM-dependent methyltransferases (SAM-MT), the C6orf211 protein showed limited sequence conservation; however, the SAM-MTs share a common structure called the SAM-MT fold13,14 and we searched the protein for this fold. By the predicting C6orf211 protein's secondary structures we identified several structures that could assemble into a SAM-MT fold (FIG. 1e) in a pattern similar to CheR (FIG. 1d). Within the SAM-MT fold is the SAM binding pocket that possesses two highly conserved sequence motifs. Alignment of the CheR motifs with full-length C6orf211 protein identified similar sequences (FIG. 1b) that showed significant evolutionary conservation (FIG. 8). Furthermore motifs I and II were positioned in the β1/αA and β2/αB loops of our hypothetical SAM-MT fold (FIG. 1e). In addition to motifs I and II, two conserved regions (II and III) have been identified in CheR and PIMT14. Alignments of the CheR regions with the C6orf211 protein identified analogous sequences with significant evolutionary conservation (FIG. 1b and FIG. 8), which, with motifs I and II, were identified in similar positions in the C6orf211 protein as CheR (FIG. 1c). These results strongly suggested that this uncharacterized protein was a methyltransferase, and expression and purification confirmed that it possessed a PCNA-dependent carboxyl methyltransferase activity (FIG. 1f & 1g). Interestingly, in addition to methyl esterifying PCNA, the C6orf211 protein or Arm1 appeared to modify itself suggesting that it may be self-regulated.
Example 16
DNA Damage Promotes PCNA Methyl Esterification
[0049] Identification of the C6orf211 gene product as Arm1, a PCNA-dependent carboxyl methyltransferase, hinted at a novel mechanism occurring in eukaryotic cells. However, the biological significance of methyl esterification was uncertain. Therefore, we used 2D-PAGE to search for PCNA methyl esterification in MCF7 breast cancer cells following DNA damage, which would be identified by a basic shift in isoelectric point (pI). In untreated cells PCNA displays a pI at or near its theoretical value of 4.510,15. But following treatment with the DNA damaging agent doxorubicin (Dox), a basic PCNA isoform (pI ˜5.6) was observed (FIG. 2a). This PCNA isoform was consistent with methyl esterification of 15+ acidic residues, and assaying the extracts for Arm1 activity showed a wave of PCNA methyl esterification that increased two hours after Dox exposure, shifted to reduced activity by 4 h, and stabilized after 6 h (FIG. 2b). This suggested that PCNA methyl esterification builds and peaks by 4 h in MCF7 cells, and by that time PCNA methyl esterification is inhibited and/or PCNA methyl esterase activity predominates over methyltransferase activity. Interestingly, increased PCNA-dependent carboxyl methyltransferase activity and the appearance of the basic PCNA isoform correlated with a >27-fold increased expression of p21.sup.WAF1/CIP1 in MCF7 cells (FIG. 2c).
[0050] Since the initial observations describing p21's interaction with PCNA and inhibition of DNA replication in response to DNA damage16, the function of the p21-PCNA interaction in the DNA damage response has remained poorly understood. In addition to DNA replication, PCNA is required for DNA repair, and p21 or the PCNA interacting peptide (PIP) of p21 have been shown to disrupt mismatch17, base excision18, and nucleotide excision repair19. Despite this inhibition of DNA repair, however, p21.sup.-/- cells display a repair defect20. It was therefore possible that PCNA methyl esterification could further our understanding of p21 in the DNA damage response, so we investigated PCNA methyl esterification and the p21-PCNA interaction (FIG. 2d). To our surprise, the p21 interaction had a direct effect on PCNA methyl esterification in untreated cell extracts and by pulling down PCNA from breast cancer cell extracts with a GST-p21 fusion we observed basic shifted PCNA isoform induced by Dox exposure. This isoform was not evident in the input extracts so it was unlikely that its appearance was the result of enrichment. Instead, these results indicated that p21 regulated PCNA methyl esterification, and that the PIP domain of p21 could produce this basic shifted isoform. Using the p21(PIP) peptide we then affinity purified PCNA (FIG. 2e) to determine if methyl esterification was indeed promoting its basic pI shift. By sequencing the affinity purified PCNA isoforms we were able to identify a consistent trend of increasing methyl esterification on the basic-shifted PCNA isoforms (table I). The positions of the methyl esters on these isoforms were also highly conserved, and appeared nearly exclusive on glutamate residues (FIG. 10). Several methyl esters were also identified at previously unrecognized positions (E124, E130, E193 and E198), and PCNA's C-terminus was found exclusively di-methyl esterified. This was an intriguing result considering our previous observations of mono-methyl esters on two separate residues of the acidic PCNA isoform10 and its involvement in the p21 interaction21. Together these data strongly supported regulation of PCNA methyl esterification by p21, and to further explore this mechanism we knocked down Arm1 expression in p53 wild-type and p53-muant cells and examined their abilities to respond to DNA damage.
Example 17
Arm1-Dependent PCNA Methyl Esterification is Linked to DNA Repair
[0051] Although the previous results support PCNA methyl esterification in the DNA damage response, the role of Arm1 in this response was still unclear. Therefore, we knocked-down Arm1 expression in MCF7(p53 wild-type) and SK-Br-3 (p53-mutant) breast cancer cells (FIG. 10). We then damaged the DNA of these cells with Dox and UV and examined PCNA mobility by 2D-PAGE (FIG. 3a). Unexpectedly, methyl esterified PCNA isoforms were evident in untreated MCF7 following Arm1 knockdown. Although the appearance of the basic isoform in the undamaged extracts could be attributed to residual Arm1 expression, it also indicated that Arm1 knockdown dysregulated PCNA modification, and in yeast Arm1 mutant cells. Additionally, posttranslational state of PCNA in Dox treated Arm1 knockdown MCF7 cells appeared drastically different to the control cells. However, the clearest results were observed following UV irradiation. A PCNA isoform indicative of methyl esterification was observed in UV treated shRNA control cells, but not in the Arm1 shRNA expressing cells (FIG. 3a) indicating that methyl esterification is also functional in response to UV. The drastic differences in PCNA's posttranslational states following Dox and UV exposures could be explained by the nature of the DNA damage generated by these agents. Although Dox generates DNA double strand breaks through topoisomerase inhibition as well as inhibition of transcription and DNA replication, it is also implicated in alkylation, cross-linking, and free-radical DNA damage22. Compared to UV damage, which predominantly generates pyrimidine dimers, Dox-generated DNA damage would necessitate multiple modes of DNA repair and PCNA's interactions with several repair factors. The appearance of numerous PCNA isoforms in the Dox treated Arm1 shRNA expressing cells could therefore be indicative of a level of control over protein-protein interactions that promote PCNA posttranslational modifications in response to numerous types of DNA damage.
[0052] In addition to p53 wild-type MCF7 cells we also examined p53-mutant SK-Br-3 cells that are unable to induce p21 expression following DNA damage. Interestingly, methyl esterified PCNA isoforms were also observed in the control cells following Dox and UV exposures and were significantly reduced in the Arm1 knockdown cells (FIG. 3a). Like MCF7 cells, loss of PCNA methyl esterification was most apparent in the SK-Br-3 Arm1 knockdown cells following UV-irradiation suggesting that PCNA methyl esterification does occur in the absence of p21 up-regulation. The role of p21 in response to UV-induced DNA damage is controversial partially due to its ability to disrupt PCNA's interaction of with nucleotide excision repair endonuclease XPG19 and p21 degradation has been shown to promote UV repair23. We therefore examined p21 expression in these cell lines following UV-irradiation (FIG. 3c). Although p21 up-regulation was not observed in SK-Br-3 cells, we did observe the accumulation of chromatin bound p21 in MCF7 cells. This suggested that p21 exerts its affects on chromatin bound PCNA in MCF7 cells in response to UV damage. PCNA chromatin stability was fairly constant in the Arm1 knockdown cells compared to the controls in response to UV with slightly higher levels of chromatin bound PCNA were observed in the Arm1 knockdown cells 2 and 6 h following UV (FIG. 3c). Arm1 levels were very low in the chromatin bound fractions suggestive of EGFR-dependent phosphorylation has been shown to protect PCNA from ubiquitin-dependent degradation and promoting chromatin stability24. It was therefore possible that ubiquitin-dependent PCNA degradation and removal from the chromatin.suggesting that Arm1 has a minimal although slightly increased levels of PCNA are slightly observed of The present on the chromatinp21 on the Additionally, contradictory results on p21's role in PCNA ubiquitylation and translesion DNA synthesis (TLS) have also been reported25,26 is also unclear and contradictory results. Arm1 could therefore be a missing factor that may help explain some of these contradictory findings when DNA damage tolerance has been observed. This suggested that there are p21-independent roles for Arm1 in the DNA damage response as well. The DNA damage-induced PCNA isoform appeared nearly unaltered in both Arm1 shRNA expressing cell lines in response to the alkylating agent MMS when compared to the controls suggesting a limited role for Arm1 in response to this type of DNA damage. However, a slower migrating potentially ubiquitylated or SUMOylated PCNA species was observed in the control and not Arm1 shRNA expressing MCF7 cells (FIG. 4a) suggesting that Arm1 could have a role in post-replication DNA repair. The absence of this slower migrating PCNA species in MMS treated SK-Br-3 cells was consistent with previous observations that p53 and p21 promoted UV-induced PCNA ubiquitylation, and that expression of the p21-PIP box was sufficient to suppress efficiency and increased fidelity of translesion DNA synthesis activity in cells26. In contrast, expression of a non-degradable p21 mutant was shown to inhibit PCNA ubiquitylation following UV damage25. Regardless of this discrepancy, p21 does appear to affect PCNA ubiquitylation, which further implicates Arm1 in post-replication DNA repair.
[0053] In addition to PCNA modification in knock-down cells, we examined cell survival following DNA damage (FIG. 4b). Interestingly, cell survival in response to DOX, MMS, and UV were significantly different in the Arm knock-down cells compared to control cells. Knock-down of Arm1 in p53 wild-type MCF7 cells led to significantly enhanced survival in response to DOX, MMS, and UV. In MCF7 cells with knocked-down Arm1 expression 47% of cells survived 0.5 mM DOX compared to 15% survival in the control cells (FIG. 4b). Similar results were consistently observed in response to UV with 39% of the Arm1 knock-down cells surviving 25 J/m2 compared to 19% in the control. And although not as dramatic, enhanced survival was also observed in response to MMS. In stark contrast to MCF7 cells, knock-down of Arm1 expression in p53-mutant SK-Br-3 cells significantly reduced survival to only 3% in response to 0.5 mM DOX compared to 18% of the control cells. These results were also consistent in UV and MMS treatments with 4% and 48% of Arm1 knock-down cells surviving 0.0025% MMS and 25 J/m2 UV compared to 12% and 82% in the control cells, respectively. These results strongly supported Arm1's function in DNA repair, and suggested that this novel signalling mechanism plays an important role in the cell's ability to properly respond to and repair DNA damage. Additionally, because ˜50% of tumours harbour p53 mutations and knock-down of Arm1 expression in p53-mutant breast tumours appeared to sensitize the cells to DNA damage, inhibition of Arm1 may ultimately allow us to more effectively target tumour cells.
DISCUSSION
[0054] We describe a novel eukaryotic protein carboxyl methyltransferase, Arm1, which specifically targets glutamic and aspartic acid residues in PCNA. We also present evidence that methyl esterification of PCNA is stimulated following exposure of cells to genotoxic stress, which is mediated, at least in part, through p21 binding. As early as 1979, the methyl esterification of glutamic acid residues in the eukaryotic proteins was reported by what was, at that time, known as protein carboxyl O-methyltransferase27. Subsequently, protein carboxyl O-methyltransferase's specificity for iso-aspartate residues and ability to facilitate protein repair led to its reassignment as protein isoaspartate methyltransferase (PIMT)28. Since that time, investigations into glutamyl methyl esterification of eukaryotic proteins have been essentially nonexistent. With the advent of proteomics and advances in modern protein mass spectrometry, the unambiguous detection of these structures on eukaryotic proteins has become possible. And since our initial observations10, at least two independent laboratories have described these structures on aspartic and glutamic acid residues in eukaryotic proteins29,30.
[0055] How methyl esterification affects PCNA's structure remains to be elucidated; but, in prokaryotic cells, chemotaxis receptor methyl esterification changes it conformations controlling protein-protein interactions effecting the cell's ability to adapt to stimuli31. Likewise, Arm1-dependent methyl esterification of PCNA may regulate its protein-protein interactions ultimately allowing the cell to adapt to genotoxic stress. Examination of the positions of methyl esterified residues on the PCNA crystal structure21 (FIG. 5) identified a concentration of these structures in the subunit interfaces indicating potential effects on trimer assembly. This is supported by observations that a glutamic acid to glycine (E113G) mutation in the subunit interface of yeast PCNA significantly affected the molecule's ability to form trimers32. Also consistent with this is the reported correlation between UV-dependent ubiquitylation and degradation of p21 and the accumulation of chromatin bound PCNA23. This further suggests that PCNA methyl esterification following p21 binding may promote PCNA disassembly from chromatin. Regardless of Arm1's mechanism, its presence appears to be required for appropriate response to DNA damage, and the data reported here may help explain some contradictory observations on p21's tumour suppressor and oncogenes functions33,34,35.
[0056] In addition to PCNA, Arm1 likely has multiple other targets and it is difficult to speculate as to whether the survival differences observed in the Arm1 knockdown cells were mediated solely through PCNA methyl esterification. However, an interaction of PCNA with ING1 was previously shown to promote UV-induced apoptosis and prevention of this interaction through either over-expression of p21 or mutation to ING1's PCNA interacting PIP-box prevented UV-induced apoptosis36. It is therefore attractive to postulate that Arm1 could regulate PCNA's interactions with, among other factors, ING. And loss of Arm1's ability to regulate PCNA's interactions may prevent the cell from effectively responding to DNA damage. Further investigations are required to determine Arm'1 exact role(s) in response to genotoxic stress, but from these results it is clear that methyl esterification of acidic protein residues is a real posttranslational mechanism that alters protein structure and function in eukaryotes.
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[0057] The following references reflect the level of knowledge in the field and are hereby incorporated by reference in their entirety. Any conflict between the teachings of these references and this specification shall be resolved in favor of the latter.
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Sequence CWU
1
1
25113PRTArtificial SequenceSynthetic - Motif I CheR (a.a. 117-129) 1Tyr
Arg Val Trp Ser Ala Ala Ala Ser Thr Gly Glu Glu 1 5
10 213PRTArtificial SequenceSynthetic - Motif I
Arm1 (a.a. 245-257) 2Thr Arg Val Tyr Ile Val Leu Asp Asn Ser Gly Phe Glu
1 5 10 39PRTArtificial
SequenceSynthetic - Motif II CheR (a.a. 152-160) 3Ala Ser Asp Ile Asp Thr
Glu Val Leu 1 5 49PRTArtificial
SequenceSynthetic - Motif II Arm1(a.a. 289-297) 4Val Ser Asp Thr Thr Ile
His Asp Phe 1 5 512PRTArtificial
SequenceSynthetic - Consensus region II CheR (a.a. 218-229) 5Tyr Thr
Val Pro Gly Pro Phe Asp Ala Ile Phe Cys 1 5
10 612PRTArtificial SequenceSynthetic - Consensus region II
Arm1 (a.a. 336-347) 6Trp Thr Leu Pro His Glu Tyr Cys Ala Met Pro Gln
1 5 10 79PRTArtificial
SequenceSynthetic - Consensus region III CheR (a.a. 251-259) 7Leu
Leu Lys Pro Asp Gly Leu Leu Phe 1 5
89PRTArtificial SequenceSynthetic - Consensus region III Arm1 (a.a.
356-364) 8Leu Gln Lys Ala His Leu Ile Leu Phe 1 5
9286PRTE.colimisc_feature(1)..(286)E.coli CheR 9Met Thr Ser Ser
Leu Pro Cys Gly Gln Thr Ser Leu Leu Leu Gln Met 1 5
10 15 Thr Glu Arg Leu Ala Leu Ser Asp Ala
His Phe Arg Arg Ile Ser Gln 20 25
30 Leu Ile Tyr Gln Arg Ala Gly Ile Val Leu Ala Asp His Lys
Arg Asp 35 40 45
Met Val Tyr Asn Arg Leu Val Arg Arg Leu Arg Ser Leu Gly Leu Thr 50
55 60 Asp Phe Gly His Tyr
Leu Asn Leu Leu Glu Ser Asn Gln His Ser Gly 65 70
75 80 Glu Trp Gln Ala Phe Ile Asn Ser Leu Thr
Thr Asn Leu Thr Ala Phe 85 90
95 Phe Arg Glu Ala His His Phe Pro Leu Leu Ala Asp His Ala Arg
Arg 100 105 110 Arg
Ser Gly Glu Tyr Arg Val Trp Ser Ala Ala Ala Ser Thr Gly Glu 115
120 125 Glu Pro Tyr Ser Ile Ala
Met Thr Leu Ala Asp Thr Leu Gly Thr Ala 130 135
140 Pro Gly Arg Trp Lys Val Phe Ala Ser Asp Ile
Asp Thr Glu Val Leu 145 150 155
160 Glu Lys Ala Arg Ser Gly Ile Tyr Arg His Glu Glu Leu Lys Asn Leu
165 170 175 Thr Pro
Gln Gln Leu Gln Arg Tyr Phe Met Arg Gly Thr Gly Pro His 180
185 190 Glu Gly Leu Val Arg Val Arg
Gln Glu Leu Ala Asn Tyr Val Asp Phe 195 200
205 Ala Pro Leu Asn Leu Leu Ala Lys Gln Tyr Thr Val
Pro Gly Pro Phe 210 215 220
Asp Ala Ile Phe Cys Arg Asn Val Met Ile Tyr Phe Asp Gln Thr Thr 225
230 235 240 Gln Gln Glu
Ile Leu Arg Arg Phe Val Pro Leu Leu Lys Pro Asp Gly 245
250 255 Leu Leu Phe Ala Gly His Ser Glu
Asn Phe Ser His Leu Glu Arg Arg 260 265
270 Phe Thr Leu Arg Gly Gln Thr Val Tyr Ala Leu Ser Lys
Asp 275 280 285 10441PRTHomo
sapiensmisc_feature(1)..(441)Human C6orf211 10Met Ala Val Val Pro Ala Ser
Leu Ser Gly Gln Asp Val Gly Ser Phe 1 5
10 15 Ala Tyr Leu Thr Ile Lys Asp Arg Ile Pro Gln
Ile Leu Thr Lys Val 20 25
30 Ile Asp Thr Leu His Arg His Lys Ser Glu Phe Phe Glu Lys His
Gly 35 40 45 Glu
Glu Gly Val Glu Ala Glu Lys Lys Ala Ile Ser Leu Leu Ser Lys 50
55 60 Leu Arg Asn Glu Leu Gln
Thr Asp Lys Pro Phe Ile Pro Leu Val Glu 65 70
75 80 Lys Phe Val Asp Thr Asp Ile Trp Asn Gln Tyr
Leu Glu Tyr Gln Gln 85 90
95 Ser Leu Leu Asn Glu Ser Asp Gly Lys Ser Arg Trp Phe Tyr Ser Pro
100 105 110 Trp Leu
Leu Val Glu Cys Tyr Met Tyr Arg Arg Ile His Glu Ala Ile 115
120 125 Ile Gln Ser Pro Pro Ile Asp
Tyr Phe Asp Val Phe Lys Glu Ser Lys 130 135
140 Glu Gln Asn Phe Tyr Gly Ser Gln Glu Ser Ile Ile
Ala Leu Cys Thr 145 150 155
160 His Leu Gln Gln Leu Ile Arg Thr Ile Glu Asp Leu Asp Glu Asn Gln
165 170 175 Leu Lys Asp
Glu Phe Phe Lys Leu Leu Gln Ile Ser Leu Trp Gly Asn 180
185 190 Lys Cys Asp Leu Ser Leu Ser Gly
Gly Glu Ser Ser Ser Gln Asn Thr 195 200
205 Asn Val Leu Asn Ser Leu Glu Asp Leu Lys Pro Phe Ile
Leu Leu Asn 210 215 220
Asp Met Glu His Leu Trp Ser Leu Leu Ser Asn Cys Lys Lys Thr Arg 225
230 235 240 Glu Lys Ala Ser
Ala Thr Arg Val Tyr Ile Val Leu Asp Asn Ser Gly 245
250 255 Phe Glu Leu Val Thr Asp Leu Ile Leu
Ala Asp Phe Leu Leu Ser Ser 260 265
270 Glu Leu Ala Thr Glu Val His Phe Tyr Gly Lys Thr Ile Pro
Trp Phe 275 280 285
Val Ser Asp Thr Thr Ile His Asp Phe Asn Trp Leu Ile Glu Gln Val 290
295 300 Lys His Ser Asn His
Lys Trp Met Ser Lys Cys Gly Ala Asp Trp Glu 305 310
315 320 Glu Tyr Ile Lys Met Gly Lys Trp Val Tyr
His Asn His Ile Phe Trp 325 330
335 Thr Leu Pro His Glu Tyr Cys Ala Met Pro Gln Val Ala Pro Asp
Leu 340 345 350 Tyr
Ala Glu Leu Gln Lys Ala His Leu Ile Leu Phe Lys Gly Asp Leu 355
360 365 Asn Tyr Arg Lys Leu Thr
Gly Asp Arg Lys Trp Glu Phe Ser Val Pro 370 375
380 Phe His Gln Ala Leu Asn Gly Phe His Pro Ala
Pro Leu Cys Thr Ile 385 390 395
400 Arg Thr Leu Lys Ala Glu Ile Gln Val Gly Leu Gln Pro Gly Gln Gly
405 410 415 Glu Gln
Leu Leu Ala Ser Glu Pro Ser Trp Trp Thr Thr Gly Lys Tyr 420
425 430 Gly Ile Phe Gln Tyr Asp Gly
Pro Leu 435 440 11227PRTHomo
sapiensmisc_feature(1)..(227)Human PIMT 11Met Ala Trp Lys Ser Gly Gly Ala
Ser His Ser Glu Leu Ile His Asn 1 5 10
15 Leu Arg Lys Asn Gly Ile Ile Lys Thr Asp Lys Val Phe
Glu Val Met 20 25 30
Leu Ala Thr Asp Arg Ser His Tyr Ala Lys Cys Asn Pro Tyr Met Asp
35 40 45 Ser Pro Gln Ser
Ile Gly Phe Gln Ala Thr Ile Ser Ala Pro His Met 50
55 60 His Ala Tyr Ala Leu Glu Leu Leu
Phe Asp Gln Leu His Glu Gly Ala 65 70
75 80 Lys Ala Leu Asp Val Gly Ser Gly Ser Gly Ile Leu
Thr Ala Cys Phe 85 90
95 Ala Arg Met Val Gly Cys Thr Gly Lys Val Ile Gly Ile Asp His Ile
100 105 110 Lys Glu Leu
Val Asp Asp Ser Ile Asn Asn Val Arg Lys Asp Asp Pro 115
120 125 Thr Leu Leu Ser Ser Gly Arg Val
Gln Leu Val Val Gly Asp Gly Arg 130 135
140 Met Gly Tyr Ala Glu Glu Ala Pro Tyr Asp Ala Ile His
Val Gly Ala 145 150 155
160 Ala Ala Pro Val Val Pro Gln Ala Leu Ile Asp Gln Leu Lys Pro Gly
165 170 175 Gly Arg Leu Ile
Leu Pro Val Gly Pro Ala Gly Gly Asn Gln Met Leu 180
185 190 Glu Gln Tyr Asp Lys Leu Gln Asp Gly
Ser Ile Lys Met Lys Pro Leu 195 200
205 Met Gly Val Ile Tyr Val Pro Leu Thr Asp Lys Glu Lys Gln
Trp Ser 210 215 220
Arg Trp Lys 225 12441PRTHomo sapiensmisc_feature(1)..(441)H.
sapiens (13375746) 12Met Ala Val Val Pro Ala Ser Leu Ser Gly Gln Asp Val
Gly Ser Phe 1 5 10 15
Ala Tyr Leu Thr Ile Lys Asp Arg Ile Pro Gln Ile Leu Thr Lys Val
20 25 30 Ile Asp Thr Leu
His Arg His Lys Ser Glu Phe Phe Glu Lys His Gly 35
40 45 Glu Glu Gly Val Glu Ala Glu Lys Lys
Ala Ile Ser Leu Leu Ser Lys 50 55
60 Leu Arg Asn Glu Leu Gln Thr Asp Lys Pro Phe Ile Pro
Leu Val Glu 65 70 75
80 Lys Phe Val Asp Thr Asp Ile Trp Asn Gln Tyr Leu Glu Tyr Gln Gln
85 90 95 Ser Leu Leu Asn
Glu Ser Asp Gly Lys Ser Arg Trp Phe Tyr Ser Pro 100
105 110 Trp Leu Leu Val Glu Cys Tyr Met Tyr
Arg Arg Ile His Glu Ala Ile 115 120
125 Ile Gln Ser Pro Pro Ile Asp Tyr Phe Asp Val Phe Lys Glu
Ser Lys 130 135 140
Glu Gln Asn Phe Tyr Gly Ser Gln Glu Ser Ile Ile Ala Leu Cys Thr 145
150 155 160 His Leu Gln Gln Leu
Ile Arg Thr Ile Glu Asp Leu Asp Glu Asn Gln 165
170 175 Leu Lys Asp Glu Phe Phe Lys Leu Leu Gln
Ile Ser Leu Trp Gly Asn 180 185
190 Lys Cys Asp Leu Ser Leu Ser Gly Gly Glu Ser Ser Ser Gln Asn
Thr 195 200 205 Asn
Val Leu Asn Ser Leu Glu Asp Leu Lys Pro Phe Ile Leu Leu Asn 210
215 220 Asp Met Glu His Leu Trp
Ser Leu Leu Ser Asn Cys Lys Lys Thr Arg 225 230
235 240 Glu Lys Ala Ser Ala Thr Arg Val Tyr Ile Val
Leu Asp Asn Ser Gly 245 250
255 Phe Glu Leu Val Thr Asp Leu Ile Leu Ala Asp Phe Leu Leu Ser Ser
260 265 270 Glu Leu
Ala Thr Glu Val His Phe Tyr Gly Lys Thr Ile Pro Trp Phe 275
280 285 Val Ser Asp Thr Thr Ile His
Asp Phe Asn Trp Leu Ile Glu Gln Val 290 295
300 Lys His Ser Asn His Lys Trp Met Ser Lys Cys Gly
Ala Asp Trp Glu 305 310 315
320 Glu Tyr Ile Lys Met Gly Lys Trp Val Tyr His Asn His Ile Phe Trp
325 330 335 Thr Leu Pro
His Glu Tyr Cys Ala Met Pro Gln Val Ala Pro Asp Leu 340
345 350 Tyr Ala Glu Leu Gln Lys Ala His
Leu Ile Leu Phe Lys Gly Asp Leu 355 360
365 Asn Tyr Arg Lys Leu Thr Gly Asp Arg Lys Trp Glu Phe
Ser Val Pro 370 375 380
Phe His Gln Ala Leu Asn Gly Phe His Pro Ala Pro Leu Cys Thr Ile 385
390 395 400 Arg Thr Leu Lys
Ala Glu Ile Gln Val Gly Leu Gln Pro Gly Gln Gly 405
410 415 Glu Gln Leu Leu Ala Ser Glu Pro Ser
Trp Trp Thr Thr Gly Lys Tyr 420 425
430 Gly Ile Phe Gln Tyr Asp Gly Pro Leu 435
440 13441PRTB. taurusmisc_feature(1)..(441)B. taurus
(134085653) 13Met Ala Gly Pro Pro Ala Ser Leu Ser Ala Arg Asp Val Gly Ser
Phe 1 5 10 15 Ala
Tyr Leu Ser Val Lys Asp Arg Ser Pro Gln Ile Leu Thr Lys Ala
20 25 30 Ile Asp Thr Leu His
Arg His Lys Ser Glu Phe Phe Glu Lys His Gly 35
40 45 Glu Lys Gly Leu Glu Ala Glu Lys Lys
Ala Ile Ser Leu Leu Ser Lys 50 55
60 Leu Arg Asn Glu Leu Gln Thr Asp Lys Pro Ile Val Pro
Leu Val Glu 65 70 75
80 Lys Phe Val Asp Thr Asp Ile Trp Asn Gln Tyr Leu Glu Tyr Gln Gln
85 90 95 Ser Leu Leu Asn
Glu Ser Asp Gly Lys Pro Arg Trp Phe Leu Ser Pro 100
105 110 Trp Leu Phe Val Glu Cys Tyr Met Tyr
Arg Arg Ile His Glu Ala Ile 115 120
125 Ile Gln Ser Pro Pro Ile Asp Asp Phe Asp Ile Phe Lys Glu
Phe Lys 130 135 140
Asp Gln Asn Phe Phe Glu Ser Gln Glu Ser Ile Ile Ala Leu Cys Thr 145
150 155 160 His Leu Gln Glu Leu
Arg Lys Thr Ile Glu Asp Leu Asp Glu Asn Gln 165
170 175 Leu Lys Asn Glu Phe Phe Lys Val Leu Gln
Ile Ser Leu Trp Gly Asn 180 185
190 Lys Cys Asp Leu Ser Leu Ser Gly Gly Glu His Ile Ser Gln Lys
Thr 195 200 205 Asn
Ile Met Asn Ser Leu Glu Asp Leu Lys Pro Phe Ile Leu Val Asn 210
215 220 Asp Met Asp Arg Leu Trp
Ser Leu Leu Ser Asn Cys Lys Lys Thr Arg 225 230
235 240 Glu Lys Glu Ser Val Thr Arg Val Asp Ile Val
Leu Asp Asn Ser Gly 245 250
255 Phe Glu Leu Ile Thr Asp Leu Val Leu Ala Asp Phe Leu Leu Ser Ser
260 265 270 Lys Leu
Ala Thr Lys Ile His Phe Tyr Gly Lys Thr Ile Pro Trp Phe 275
280 285 Val Ser Asp Thr Thr Leu His
Asp Phe Asn Trp Ile Ile Lys Gln Leu 290 295
300 Lys His Ser Asn Asn Lys Trp Val Ser Gln Cys Gly
Val Asp Trp Glu 305 310 315
320 Asp His Val Lys Thr Gly Arg Trp Val Tyr Leu Asp His Ile Phe Trp
325 330 335 Thr Leu Pro
His Glu Phe Ser Ala Met Ser Gln Val Ala Pro Asp Leu 340
345 350 His Ala Ala Leu Gln Lys Ala His
Leu Ile Phe Phe Lys Gly Asp Leu 355 360
365 Asn Tyr Arg Lys Leu Thr Gly Asp Arg Arg Trp Glu Phe
Thr Val Pro 370 375 380
Phe His Glu Ala Leu Ser Gly Phe His Pro Ala Pro Leu Cys Ser Ile 385
390 395 400 Arg Thr Leu Lys
Ala Glu Val Gln Val Gly Leu Gln Pro Gly Gln Gly 405
410 415 Glu Gln Leu Thr Ala Ser Glu Pro Asn
Trp Leu Thr Ala Gly Lys Tyr 420 425
430 Gly Val Phe Gln Phe Asp Gly Pro Leu 435
440 14439PRTM. musculusmisc_feature(1)..(439)M. musculus
(90093343) 14Met Ala Glu Ser Pro Ala Phe Leu Ser Ala Lys Asp Glu Gly Ser
Phe 1 5 10 15 Ala
Tyr Leu Thr Ile Lys Asp Arg Thr Pro Gln Ile Leu Thr Lys Val
20 25 30 Ile Asp Thr Leu His
Arg His Lys Ser Glu Phe Phe Glu Lys His Gly 35
40 45 Glu Glu Gly Ile Glu Ala Glu Lys Lys
Ala Ile Ser Leu Leu Ser Lys 50 55
60 Leu Arg Asn Glu Leu Gln Thr Asp Lys Pro Ile Thr Pro
Leu Val Asp 65 70 75
80 Lys Cys Val Asp Thr His Ile Trp Asn Gln Tyr Leu Glu Tyr Gln Arg
85 90 95 Ser Leu Leu Asn
Glu Gly Asp Gly Glu Pro Arg Trp Phe Phe Ser Pro 100
105 110 Trp Leu Phe Val Glu Cys Tyr Met Tyr
Arg Arg Ile His Glu Ala Ile 115 120
125 Met Gln Ser Pro Pro Ile His Asp Phe Asp Val Phe Lys Glu
Ser Lys 130 135 140
Glu Glu Asn Phe Phe Glu Ser Gln Gly Ser Ile Asp Ala Leu Cys Ser 145
150 155 160 His Leu Leu Gln Leu
Lys Pro Val Lys Gly Leu Arg Glu Glu Gln Ile 165
170 175 Gln Asp Glu Phe Phe Lys Leu Leu Gln Ile
Ser Leu Trp Gly Asn Lys 180 185
190 Cys Asp Leu Ser Leu Ser Gly Gly Glu Ser Ser Ser Gln Lys Ala
Asn 195 200 205 Ile
Ile Asn Cys Leu Gln Asp Leu Lys Pro Phe Ile Leu Ile Asn Asp 210
215 220 Thr Glu Ser Leu Trp Ala
Leu Leu Ser Lys Leu Lys Lys Thr Val Glu 225 230
235 240 Thr Pro Val Val Arg Val Asp Ile Val Leu Asp
Asn Ser Gly Phe Glu 245 250
255 Leu Ile Thr Asp Leu Val Leu Ala Asp Phe Leu Phe Ser Ser Glu Leu
260 265 270 Ala Thr
Glu Ile His Phe His Gly Lys Ser Ile Pro Trp Phe Val Ser 275
280 285 Asp Val Thr Glu His Asp Phe
Asn Trp Ile Val Glu His Met Lys Ser 290 295
300 Ser Asn Leu Glu Ser Met Ser Thr Cys Gly Ala Cys
Trp Glu Ala Tyr 305 310 315
320 Ala Arg Met Gly Arg Trp Ala Tyr His Asp His Ala Phe Trp Thr Leu
325 330 335 Pro His Pro
Tyr Cys Val Met Pro Gln Val Ala Pro Asp Leu Tyr Ala 340
345 350 Glu Leu Gln Lys Ala His Leu Ile
Leu Phe Lys Gly Asp Leu Asn Tyr 355 360
365 Arg Lys Leu Met Gly Asp Arg Lys Trp Lys Phe Thr Phe
Pro Phe His 370 375 380
Gln Ala Leu Ser Gly Phe His Pro Ala Pro Leu Cys Ser Ile Arg Thr 385
390 395 400 Leu Lys Cys Glu
Leu Gln Val Gly Leu Gln Pro Gly Gln Ala Glu Gln 405
410 415 Leu Thr Ala Ser Asp Pro His Trp Leu
Thr Thr Gly Arg Tyr Gly Ile 420 425
430 Leu Gln Phe Asp Gly Pro Leu 435
15448PRTD. reriomisc_feature(1)..(448)D. rerio (62122847) 15Met Glu Ala
Glu Gly Met Leu Pro Pro Gln Ser Leu Ser Ala Lys Phe 1 5
10 15 Glu Gly Ser Phe Ala Tyr Leu Thr
Val Arg Asp Arg Leu Pro Thr Ile 20 25
30 Leu Thr Lys Val Val Asp Thr Leu His Arg Asn Lys Asp
Asn Phe Tyr 35 40 45
Lys Glu Tyr Gly Glu Glu Gly Thr Glu Ala Glu Lys Arg Ala Ile Ser 50
55 60 Phe Leu Ser Arg
Leu Arg Asn Glu Leu Gln Thr Asp Lys Pro Val Leu 65 70
75 80 Ala Leu Thr Asp Asn Ala Glu Asp Thr
Gln Ala Trp Asn Glu Tyr Met 85 90
95 Glu Arg Gln Gln Asp Leu Met Glu Asn Gly Gln Leu Val Ser
Trp Phe 100 105 110
Lys Ser Pro Trp Leu Tyr Val Glu Cys Tyr Met Tyr Arg Arg Ile Gln
115 120 125 Glu Ala Leu Tyr
Met Asn Pro Pro Met His Asn Phe Asp Pro Phe Lys 130
135 140 Glu Gly Lys Thr Gln Ser Tyr Phe
Glu Ser Gln Gln Ala Ile Lys Tyr 145 150
155 160 Leu Cys Thr Tyr Leu Gln Glu Leu Ile Thr Asn Met
Glu Asn Met Thr 165 170
175 Glu Ile Gln Leu Arg Glu Asn Phe Leu Lys Leu Ile Gln Val Ser Leu
180 185 190 Trp Gly Asn
Lys Cys Asp Leu Ser Ile Ser Ala Gly Gln Asp Asn Ser 195
200 205 Gln Lys Leu Ser Pro Ile Asp Ser
Leu Pro Asp Leu Gln Arg Phe Ile 210 215
220 Leu Val Asp Asp Ser Ser Met Val Trp Ser Thr Leu Val
Ala Ser Gln 225 230 235
240 Gly Ser Arg Ser Ser Gly Met Lys His Ala Arg Val Asp Ile Ile Leu
245 250 255 Asp Asn Ala Gly
Phe Glu Leu Val Thr Asp Leu Val Leu Ala Asp Phe 260
265 270 Leu Ile Ser Ser Gly Leu Ala Lys Gln
Ile Arg Phe His Gly Lys Ser 275 280
285 Ile Pro Trp Phe Val Ser Asp Val Thr Lys Gln Asp Phe Glu
Trp Thr 290 295 300
Ile Lys Gln Thr Met Ala Ala Asn His Lys Trp Met Ser Ala Ser Gly 305
310 315 320 Val Gln Trp Lys His
Phe Met Lys Glu Gly Thr Trp Ser Tyr His Asp 325
330 335 His Pro Phe Trp Thr Leu Pro His Glu Phe
Cys Asp Met Thr Val Asp 340 345
350 Ala Ala Asn Leu Tyr Ser Thr Leu Gln Thr Ser Asp Leu Ile Leu
Phe 355 360 365 Lys
Gly Asp Leu Asn Tyr Arg Lys Leu Thr Gly Asp Arg Lys Trp Glu 370
375 380 His Thr Val Arg Phe Asp
Gln Ala Leu Arg Gly Phe Gln Pro Ala Pro 385 390
395 400 Leu Cys Ser Leu Arg Thr Leu Lys Ala Asp Val
Gln Val Gly Leu Gln 405 410
415 Ala Gly His Ala Glu Lys Leu Ser Thr Gln Asp Pro Asp Trp Met Thr
420 425 430 Asn Gly
Lys Tyr Ala Val Val Gln Phe Ser Ser Pro His Arg Glu Gln 435
440 445 16436PRTD.
melanogastermisc_feature(1)..(436)D. melanogaster (24657549) 16Met Gly
Ser Glu Thr Asp Phe Asp Ala Lys Asn Gly Ile Val Asp Gly 1 5
10 15 Pro Thr Pro Pro His Thr Glu
Leu Ala Ala Leu Tyr Lys Gln Ser Phe 20 25
30 Ala Tyr Tyr Thr Phe Arg Val Arg Leu Pro Ser Thr
Leu Ala Thr Ile 35 40 45
Ala Asp Ser Leu Val Lys Asp Lys Asp Val Leu Leu Ala Thr Tyr Gly
50 55 60 Ala Ala Ala
Glu Ala Asp Ile Glu Gln Thr Thr Lys Glu Val Arg Gln 65
70 75 80 Leu Arg Asp Asp Ile Leu Ser
Asn Gly Pro Leu Leu Pro Phe Gly Glu 85
90 95 Asn Asp Ser Asp Ser Glu Val Trp Asn Ala Phe
Leu Glu Lys Leu Pro 100 105
110 Lys Glu Lys Arg Thr Tyr Phe Ser Val Cys Trp Leu Tyr Ala Glu
Cys 115 120 125 Tyr
Met Tyr Arg Lys Ile Ser Ser Ile Phe Arg Ala Thr Ala His Leu 130
135 140 Ala Ala Tyr Asp Tyr Phe
Ser Gln Gln Lys Gln Thr Ala Thr Lys Leu 145 150
155 160 Ser Val Asp Ala Met Leu Ala Val Ala Lys Ala
Thr Arg His Asn Glu 165 170
175 Arg Asn Ser Asp Thr Phe Arg Gln Leu Ile Lys Leu Asn Leu Trp Gly
180 185 190 Asn Arg
Cys Asp Leu Ser Ile Thr Ser Gly Lys Gln Val Lys Pro Thr 195
200 205 Gly Asn Ala Phe Asp Gln Val
Thr Asp Leu Glu Glu Lys Leu Leu Ile 210 215
220 Asp Gly Thr Ala Glu Val Trp Lys Ala Leu Asp Gly
Ala Ser Gly Glu 225 230 235
240 Gly Ile Val Asp Ile Ile Phe Asp Asn Ala Gly Tyr Glu Leu Tyr Thr
245 250 255 Asp Leu Ile
Leu Ala Glu Tyr Ile Ile Asp Lys Gly Leu Ala Ala Lys 260
265 270 Val Arg Phe Asn Pro Lys Ala Ile
Pro Trp Phe Ile Ser Asp Val Met 275 280
285 Glu His Asp Phe His Trp Ala Leu Gln Phe Leu Ala Asp
His Pro Asp 290 295 300
Pro Val Leu Ser Glu Val Gly Lys Lys Trp Gln Arg Leu Thr Thr Glu 305
310 315 320 Gly Lys Phe Glu
Leu Ser Pro Leu Glu His Phe Trp Thr Ser Pro Tyr 325
330 335 Glu Phe Tyr Arg Met Pro Glu Val Asn
Pro Ser Leu Tyr Asp Arg Leu 340 345
350 Lys Glu Ala Gln Leu Val Ile Phe Lys Gly Asp Leu Asn Tyr
Arg Lys 355 360 365
Leu Leu Gly Asp Phe Ser Trp Asp Ser Thr Glu Ser Phe Glu Thr Cys 370
375 380 Leu Arg Gly Phe Arg
Pro Ser Asn Leu Cys Thr Leu Arg Thr Ile Lys 385 390
395 400 Ala Asp Leu Ile Cys Gly Leu Gly Ala Gly
Val Ala Asp Gln Leu Phe 405 410
415 Ala Lys Asp Lys Glu Trp Met Leu Thr Gly Glu Tyr Gly Val Ile
Gln 420 425 430 Phe
Ala Ser Lys 435 17450PRTC. elegansmisc_feature(1)..(450)C.
elegans (17560374) 17Met Glu Asn Ala Asp Glu Tyr Asp His Leu Ala Pro Lys
Leu Arg Gly 1 5 10 15
Lys Lys Glu Gly Thr Phe Ala Tyr Tyr Thr Val Arg Asp Arg Trp Pro
20 25 30 Lys Ile Val Thr
Gly Leu Val Asp Gln Leu Ala Gln Lys Arg Ala Ser 35
40 45 Leu Ile Glu Lys Tyr Gly Ser Glu Val
Glu Ser Asp Ile Ala Ala Ile 50 55
60 Leu Glu Val Phe Ser Lys Leu Arg Tyr Glu Ile Met Thr
Asp Lys Pro 65 70 75
80 Leu Cys Asn Leu Met Asp Thr Gln Leu Asp Ser Glu Met Trp Arg Asn
85 90 95 Leu Leu Ser Asp
Met Arg Thr Ala Ala Met Pro Asp Glu Val Glu Asp 100
105 110 Leu Thr Phe Phe Lys Gly Pro Trp Leu
Phe Val Glu Cys Trp Leu Tyr 115 120
125 Arg Phe Ile Trp Ser Thr Phe Ala Lys Thr Ile Arg Leu Ser
Glu Tyr 130 135 140
Asp Tyr Phe Gln Asp Ser Lys Arg Lys Asn Phe Leu Asp His Leu Pro 145
150 155 160 Gln Ile Glu Glu Ser
Ala Ala Phe Ile Asn Lys Ile Ser Ala Lys Asp 165
170 175 Ala Pro Val His Glu Leu Phe Gly Ile Asn
Thr Ile Leu Lys Met Ser 180 185
190 Leu Trp Gly Asn Arg Ala Asp Met Ser Leu Thr Gly Gly Asp Asp
His 195 200 205 Thr
Leu Ala Met Ser Ser Met Ser Ala Ser Ser Lys Leu Ala Asp Phe 210
215 220 Val Leu Ile Asp Asp Val
Asn Asp Met Ile Val Lys Val Leu Gly Pro 225 230
235 240 Leu Lys Ile Asn Ala Asn His Glu Thr Asn Arg
Arg Ile Asp Ile Ile 245 250
255 Leu Asp Asn Ser Gly Val Glu Leu Thr Gly Asp Leu Ile Val Ala Glu
260 265 270 Phe Phe
Ile Ser Arg Gly Phe Ala Asp Lys Val Val Ile His Gly Lys 275
280 285 Ala Ile Pro Trp Phe Val Ser
Asp Val Thr Lys Pro Asp Leu Asp Trp 290 295
300 Thr Ile Glu Gln Leu Lys Asn Gly Glu Asn Ile Gly
Glu Glu Ser Arg 305 310 315
320 Ala Leu Gly Glu Lys Leu Glu Lys Arg Met Lys Ser Gly Gln Ile Val
325 330 335 Tyr Gln Asp
His Leu Phe Trp Ile Ser Pro His Ala Tyr Tyr Ala Met 340
345 350 Glu Lys Glu Ala Arg Asp Leu Tyr
Asp Asp Leu Lys Asn Ser Ser Leu 355 360
365 Ile Ile Phe Lys Gly Asp Leu Asn Tyr Arg Lys Leu Val
Gly Asp Arg 370 375 380
Asp Trp Asp Leu Asp Thr Ser Phe Lys Thr Ala Cys Arg Gly Phe Ala 385
390 395 400 Pro Cys Pro Phe
Met Ala Leu Arg Thr Leu Lys Ala Glu Thr Val Ala 405
410 415 Gly Leu Ser Glu Glu Ser Ile Ala Ile
Leu Leu Glu Lys Phe Glu Glu 420 425
430 Asp Asn Thr Trp Met Thr Ser Gly Glu Tyr Ala Val Cys Gln
Leu Gly 435 440 445
Gly Ile 450 18442PRTS. pombemisc_feature(1)..(442)S. pombe (19075471)
18Met Gly Leu Lys Leu Leu His Pro Pro Lys Pro Tyr Ala Met Thr Ser 1
5 10 15 Asp Pro Glu Ser
Tyr Ala Ser Val Cys Val Met Lys Lys Trp Pro Ile 20
25 30 Ile Ala Thr Asn Val Ile Asp Glu Val
Ser Arg Asn Ile Ser Lys Ala 35 40
45 Leu Glu Ala Gly Met Ser Asp Lys Ala Ala Tyr Val Thr Gln
Gly Lys 50 55 60
Glu Ile Ile Ser Leu Leu Asn Gln Leu Lys Tyr Asp Leu Gln His Asn 65
70 75 80 Arg Pro Leu Lys Pro
Leu Val Gly Gln Gly Pro Asp Ile Asp Asp Tyr 85
90 95 Asn Glu Glu Leu Glu Gln Val Gly Pro Leu
Thr Trp Gly Asp Ala Pro 100 105
110 Trp Leu Tyr Ala Gly Cys Tyr Phe Tyr Arg Ile Met Ser Leu Phe
Phe 115 120 125 Gln
Ala Arg Ser Glu Trp Asn Arg His Asp Pro Phe Phe Glu Gln Lys 130
135 140 Asp Phe Thr Leu Arg Ser
Ser Lys Ser Ala Ile Glu Glu Phe Ala Lys 145 150
155 160 Arg Tyr Val His Leu Asn Ser Glu Leu Ala Ser
Ile Gln Glu Asn Lys 165 170
175 Asp Asp Lys Ala Ala Tyr Met Ile Phe Val Glu Met Ala Glu Ile Ser
180 185 190 Leu Trp
Gly Asn Ala Ile Asp Leu Gly Leu Leu Val Asn Ala Thr Tyr 195
200 205 Glu Gln Leu Gln Ser Leu Gln
Gly Gln Lys Ala Val Glu Glu Ser Gln 210 215
220 Lys Asn Ile Leu Val Asn Asp Phe Pro Lys Ile Trp
Ser Lys Leu Ser 225 230 235
240 Lys Val Arg His Gly Arg Ile Asp Phe Val Leu Asp Asn Ala Gly Phe
245 250 255 Glu Leu Phe
Val Asp Leu Leu Phe Ala Thr Tyr Leu Leu Lys Thr Glu 260
265 270 Ile Ala Glu Thr Ile Ile Leu His
Pro Lys Asp Val Pro Trp Phe Val 275 280
285 Ser Asp Val Leu Val Asn Asp Ile Pro His Leu Phe Asn
Ser Leu Thr 290 295 300
Ser Tyr Phe Ser Gly Glu Gly Val Gln Lys Leu Ala Ser Asp Leu Ala 305
310 315 320 Glu Phe His Ala
Glu Gly Lys Ile Val Ile Arg Pro Asn Pro Phe Trp 325
330 335 Thr Thr Ala His Tyr Phe Gly Arg Leu
Pro Asp Val Ala Pro Lys Leu 340 345
350 Leu Ser Asp Leu Glu Gln Ser Asp Met Val Ile Phe Lys Gly
Asp Leu 355 360 365
Asn Phe Arg Lys Leu Thr Gly Asp Cys Glu Trp Pro His Thr Thr Pro 370
375 380 Phe Ala Glu Ala Leu
Gly Pro Ile Ala Gly Lys Phe Asn Ile Leu Ala 385 390
395 400 Leu Arg Thr Ile Lys Ala Asp Val Val Val
Gly Leu Gly Lys Gly Val 405 410
415 Tyr Glu Glu Ile Ala Lys Asp Asn Pro His Trp Glu Arg Thr Gly
Lys 420 425 430 Tyr
Ala Val Val Glu Phe Cys Pro Lys Asp 435 440
19470PRTS. cerevisiaemisc_feature(1)..(470)S. cerevisiae (6323669)
19Met Thr Ile Pro Gly Arg Phe Met Thr Ile Asp Lys Gly Thr Phe Gly 1
5 10 15 Glu Tyr Thr Ala
Ser Thr Arg Trp Pro Ile Ile Ile Gln Asn Ala Ile 20
25 30 Asp Asp Leu Ser Lys His Gln Glu Thr
Glu Lys Ser Asn Gly Thr Lys 35 40
45 Phe Glu Gln Gly Glu Val Ile Lys Lys Glu Leu Lys Glu Phe
Arg Gln 50 55 60
Glu Ile Ile Asp Arg Val Pro Leu Arg Pro Phe Thr Glu Glu Glu Ile 65
70 75 80 Lys Ile Ala Asn Val
Pro Leu Ser Phe Asn Glu Tyr Leu Lys Lys His 85
90 95 Pro Glu Val Asn Trp Gly Ala Val Glu Trp
Leu Phe Ser Glu Val Tyr 100 105
110 Leu Tyr Arg Arg Val Asn Val Leu Phe Gln Arg Gln Cys Glu Trp
Ala 115 120 125 Lys
Phe Asp Ile Phe Asn Arg Leu Lys Gln Ser Thr Phe Glu Ser Ser 130
135 140 Phe Tyr Gly Val Val Glu
Leu Ala Leu Arg Tyr Glu Asn Leu Leu Pro 145 150
155 160 Gln Leu Arg Glu Met Lys Gln Asn Pro Gly Asn
Glu Ile Asp Asp Ile 165 170
175 Leu Lys Val Leu Phe Lys Glu Phe Ile Glu Ile Ser Leu Trp Gly Asn
180 185 190 Ala Thr
Asp Leu Ser Leu Leu Thr Asn Ala Thr Leu Glu Asp Ile Lys 195
200 205 Ser Ile Gln Gly Ala Lys Ala
Arg Ala Ala Ser Glu Ser Lys Ile Val 210 215
220 Val Asn Asp Thr Glu Lys Ala Trp Glu Val Leu Thr
Lys Ala Arg Ala 225 230 235
240 Asp Ala Asn Ser Arg Glu Ile Arg Val Asp Phe Val Leu Asp Asn Ser
245 250 255 Gly Phe Glu
Leu Tyr Ala Asp Leu Met Leu Ala Ala Phe Leu Leu Gln 260
265 270 Ser Gly Leu Ala Thr Lys Cys Ile
Phe His Ala Lys Asp Ile Pro Tyr 275 280
285 Met Val Ser Asp Val Met Leu Lys Asp Phe Asp Ile Leu
Val His Asp 290 295 300
Leu Arg Asp Arg Glu Phe Phe Pro Ser Gly Glu Pro Ser Thr Lys Glu 305
310 315 320 Ser Arg Ala Leu
Asp Leu Phe Ala Gly Glu Met Glu Lys Phe Val Ser 325
330 335 Ser Gly Lys Ile Glu Phe Arg Glu Asp
Ser Phe Trp Thr Thr Glu Leu 340 345
350 Asp Tyr Trp Asn Leu Asp Ala Asn Glu Thr Lys Tyr His Gly
Ser Ile 355 360 365
Leu His Lys Asp Leu Gln Lys Ser Asn Leu Val Ile Phe Lys Gly Asp 370
375 380 Leu Asn Tyr Arg Lys
Leu Thr Gly Asp Arg Lys Trp Pro Arg Thr Thr 385 390
395 400 Lys Trp Glu Thr Ala Ile Gly Pro Leu Ala
Thr Asn Gly Ile Thr Ser 405 410
415 Leu Ser Leu Arg Thr Cys Lys Ala Asp Val Gln Val Ala Leu Pro
Glu 420 425 430 Gly
Leu Asp Ala Lys Leu Ser Gln Glu Trp Glu Lys Glu Asn Pro Gly 435
440 445 Arg Gly Ser Trp Trp Cys
Cys Ser Gly Lys Trp Ala Val Ile Cys Phe 450 455
460 Cys Ser Gly Ile His Lys 465
470 20261PRTArtificial SequenceSynthetic - Spot 1 20Met Phe Glu Ala Arg
Leu Val Gln Gly Ser Ile Leu Lys Lys Val Leu 1 5
10 15 Glu Ala Leu Lys Asp Leu Ile Asn Glu Ala
Cys Trp Asp Ile Ser Ser 20 25
30 Ser Gly Val Asn Leu Gln Ser Met Asp Ser Ser His Val Ser Leu
Val 35 40 45 Gln
Leu Thr Leu Arg Ser Glu Gly Phe Asp Thr Tyr Arg Cys Asp Arg 50
55 60 Asn Leu Ala Met Gly Val
Asn Leu Thr Ser Met Ser Lys Ile Leu Lys 65 70
75 80 Cys Ala Gly Asn Glu Asp Ile Ile Thr Leu Arg
Ala Glu Asp Asn Ala 85 90
95 Asp Thr Leu Ala Leu Val Phe Glu Ala Pro Asn Gln Glu Lys Val Ser
100 105 110 Asp Tyr
Glu Met Lys Leu Met Asp Leu Asp Val Glu Gln Leu Gly Ile 115
120 125 Pro Glu Gln Glu Tyr Ser Cys
Val Val Lys Met Pro Ser Gly Glu Phe 130 135
140 Ala Arg Ile Cys Arg Asp Leu Ser His Ile Gly Asp
Ala Val Val Ile 145 150 155
160 Ser Cys Ala Lys Asp Gly Val Lys Phe Ser Ala Ser Gly Glu Leu Gly
165 170 175 Asn Gly Asn
Ile Lys Leu Ser Gln Thr Ser Asn Val Asp Lys Glu Glu 180
185 190 Glu Ala Val Thr Ile Glu Met Asn
Glu Pro Val Gln Leu Thr Phe Ala 195 200
205 Leu Arg Tyr Leu Asn Phe Phe Thr Lys Ala Thr Pro Leu
Ser Ser Thr 210 215 220
Val Thr Leu Ser Met Ser Ala Asp Val Pro Leu Val Val Glu Tyr Lys 225
230 235 240 Ile Ala Asp Met
Gly His Leu Lys Tyr Tyr Leu Ala Pro Lys Ile Glu 245
250 255 Asp Glu Glu Gly Ser 260
21261PRTArtificial SequenceSynthetic - Spot 2 21Met Phe Glu Ala Arg
Leu Val Gln Gly Ser Ile Leu Lys Lys Val Leu 1 5
10 15 Glu Ala Leu Lys Asp Leu Ile Asn Glu Ala
Cys Trp Asp Ile Ser Ser 20 25
30 Ser Gly Val Asn Leu Gln Ser Met Asp Ser Ser His Val Ser Leu
Val 35 40 45 Gln
Leu Thr Leu Arg Ser Glu Gly Phe Asp Thr Tyr Arg Cys Asp Arg 50
55 60 Asn Leu Ala Met Gly Val
Asn Leu Thr Ser Met Ser Lys Ile Leu Lys 65 70
75 80 Cys Ala Gly Asn Glu Asp Ile Ile Thr Leu Arg
Ala Glu Asp Asn Ala 85 90
95 Asp Thr Leu Ala Leu Val Phe Glu Ala Pro Asn Gln Glu Lys Val Ser
100 105 110 Asp Tyr
Glu Met Lys Leu Met Asp Leu Asp Val Glu Gln Leu Gly Ile 115
120 125 Pro Glu Gln Glu Tyr Ser Cys
Val Val Lys Met Pro Ser Gly Glu Phe 130 135
140 Ala Arg Ile Cys Arg Asp Leu Ser His Ile Gly Asp
Ala Val Val Ile 145 150 155
160 Ser Cys Ala Lys Asp Gly Val Lys Phe Ser Ala Ser Gly Glu Leu Gly
165 170 175 Asn Gly Asn
Ile Lys Leu Ser Gln Thr Ser Asn Val Asp Lys Glu Glu 180
185 190 Glu Ala Val Thr Ile Glu Met Asn
Glu Pro Val Gln Leu Thr Phe Ala 195 200
205 Leu Arg Tyr Leu Asn Phe Phe Thr Lys Ala Thr Pro Leu
Ser Ser Thr 210 215 220
Val Thr Leu Ser Met Ser Ala Asp Val Pro Leu Val Val Glu Tyr Lys 225
230 235 240 Ile Ala Asp Met
Gly His Leu Lys Tyr Tyr Leu Ala Pro Lys Ile Glu 245
250 255 Asp Glu Glu Gly Ser 260
22261PRTArtificial SequenceSynthetic - Spot 3 22Met Phe Glu Ala Arg
Leu Val Gln Gly Ser Ile Leu Lys Lys Val Leu 1 5
10 15 Glu Ala Leu Lys Asp Leu Ile Asn Glu Ala
Cys Trp Asp Ile Ser Ser 20 25
30 Ser Gly Val Asn Leu Gln Ser Met Asp Ser Ser His Val Ser Leu
Val 35 40 45 Gln
Leu Thr Leu Arg Ser Glu Gly Phe Asp Thr Tyr Arg Cys Asp Arg 50
55 60 Asn Leu Ala Met Gly Val
Asn Leu Thr Ser Met Ser Lys Ile Leu Lys 65 70
75 80 Cys Ala Gly Asn Glu Asp Ile Ile Thr Leu Arg
Ala Glu Asp Asn Ala 85 90
95 Asp Thr Leu Ala Leu Val Phe Glu Ala Pro Asn Gln Glu Lys Val Ser
100 105 110 Asp Tyr
Glu Met Lys Leu Met Asp Leu Asp Val Glu Gln Leu Gly Ile 115
120 125 Pro Glu Gln Glu Tyr Ser Cys
Val Val Lys Met Pro Ser Gly Glu Phe 130 135
140 Ala Arg Ile Cys Arg Asp Leu Ser His Ile Gly Asp
Ala Val Val Ile 145 150 155
160 Ser Cys Ala Lys Asp Gly Val Lys Phe Ser Ala Ser Gly Glu Leu Gly
165 170 175 Asn Gly Asn
Ile Lys Leu Ser Gln Thr Ser Asn Val Asp Lys Glu Glu 180
185 190 Glu Ala Val Thr Ile Glu Met Asn
Glu Pro Val Gln Leu Thr Phe Ala 195 200
205 Leu Arg Tyr Leu Asn Phe Phe Thr Lys Ala Thr Pro Leu
Ser Ser Thr 210 215 220
Val Thr Leu Ser Met Ser Ala Asp Val Pro Leu Val Val Glu Tyr Lys 225
230 235 240 Ile Ala Asp Met
Gly His Leu Lys Tyr Tyr Leu Ala Pro Lys Ile Glu 245
250 255 Asp Glu Glu Gly Ser 260
23261PRTArtificial SequenceSynthetic - Spot 4 23Met Phe Glu Ala Arg
Leu Val Gln Gly Ser Ile Leu Lys Lys Val Leu 1 5
10 15 Glu Ala Leu Lys Asp Leu Ile Asn Glu Ala
Cys Trp Asp Ile Ser Ser 20 25
30 Ser Gly Val Asn Leu Gln Ser Met Asp Ser Ser His Val Ser Leu
Val 35 40 45 Gln
Leu Thr Leu Arg Ser Glu Gly Phe Asp Thr Tyr Arg Cys Asp Arg 50
55 60 Asn Leu Ala Met Gly Val
Asn Leu Thr Ser Met Ser Lys Ile Leu Lys 65 70
75 80 Cys Ala Gly Asn Glu Asp Ile Ile Thr Leu Arg
Ala Glu Asp Asn Ala 85 90
95 Asp Thr Leu Ala Leu Val Phe Glu Ala Pro Asn Gln Glu Lys Val Ser
100 105 110 Asp Tyr
Glu Met Lys Leu Met Asp Leu Asp Val Glu Gln Leu Gly Ile 115
120 125 Pro Glu Gln Glu Tyr Ser Cys
Val Val Lys Met Pro Ser Gly Glu Phe 130 135
140 Ala Arg Ile Cys Arg Asp Leu Ser His Ile Gly Asp
Ala Val Val Ile 145 150 155
160 Ser Cys Ala Lys Asp Gly Val Lys Phe Ser Ala Ser Gly Glu Leu Gly
165 170 175 Asn Gly Asn
Ile Lys Leu Ser Gln Thr Ser Asn Val Asp Lys Glu Glu 180
185 190 Glu Ala Val Thr Ile Glu Met Asn
Glu Pro Val Gln Leu Thr Phe Ala 195 200
205 Leu Arg Tyr Leu Asn Phe Phe Thr Lys Ala Thr Pro Leu
Ser Ser Thr 210 215 220
Val Thr Leu Ser Met Ser Ala Asp Val Pro Leu Val Val Glu Tyr Lys 225
230 235 240 Ile Ala Asp Met
Gly His Leu Lys Tyr Tyr Leu Ala Pro Lys Ile Glu 245
250 255 Asp Glu Glu Gly Ser 260
24261PRTArtificial SequenceSynthetic - Spot 5 24Met Phe Glu Ala Arg
Leu Val Gln Gly Ser Ile Leu Lys Lys Val Leu 1 5
10 15 Glu Ala Leu Lys Asp Leu Ile Asn Glu Ala
Cys Trp Asp Ile Ser Ser 20 25
30 Ser Gly Val Asn Leu Gln Ser Met Asp Ser Ser His Val Ser Leu
Val 35 40 45 Gln
Leu Thr Leu Arg Ser Glu Gly Phe Asp Thr Tyr Arg Cys Asp Arg 50
55 60 Asn Leu Ala Met Gly Val
Asn Leu Thr Ser Met Ser Lys Ile Leu Lys 65 70
75 80 Cys Ala Gly Asn Glu Asp Ile Ile Thr Leu Arg
Ala Glu Asp Asn Ala 85 90
95 Asp Thr Leu Ala Leu Val Phe Glu Ala Pro Asn Gln Glu Lys Val Ser
100 105 110 Asp Tyr
Glu Met Lys Leu Met Asp Leu Asp Val Glu Gln Leu Gly Ile 115
120 125 Pro Glu Gln Glu Tyr Ser Cys
Val Val Lys Met Pro Ser Gly Glu Phe 130 135
140 Ala Arg Ile Cys Arg Asp Leu Ser His Ile Gly Asp
Ala Val Val Ile 145 150 155
160 Ser Cys Ala Lys Asp Gly Val Lys Phe Ser Ala Ser Gly Glu Leu Gly
165 170 175 Asn Gly Asn
Ile Lys Leu Ser Gln Thr Ser Asn Val Asp Lys Glu Glu 180
185 190 Glu Ala Val Thr Ile Glu Met Asn
Glu Pro Val Gln Leu Thr Phe Ala 195 200
205 Leu Arg Tyr Leu Asn Phe Phe Thr Lys Ala Thr Pro Leu
Ser Ser Thr 210 215 220
Val Thr Leu Ser Met Ser Ala Asp Val Pro Leu Val Val Glu Tyr Lys 225
230 235 240 Ile Ala Asp Met
Gly His Leu Lys Tyr Tyr Leu Ala Pro Lys Ile Glu 245
250 255 Asp Glu Glu Gly Ser 260
25261PRTArtificial SequenceSynthetic - Spot 6 25Met Phe Glu Ala Arg
Leu Val Gln Gly Ser Ile Leu Lys Lys Val Leu 1 5
10 15 Glu Ala Leu Lys Asp Leu Ile Asn Glu Ala
Cys Trp Asp Ile Ser Ser 20 25
30 Ser Gly Val Asn Leu Gln Ser Met Asp Ser Ser His Val Ser Leu
Val 35 40 45 Gln
Leu Thr Leu Arg Ser Glu Gly Phe Asp Thr Tyr Arg Cys Asp Arg 50
55 60 Asn Leu Ala Met Gly Val
Asn Leu Thr Ser Met Ser Lys Ile Leu Lys 65 70
75 80 Cys Ala Gly Asn Glu Asp Ile Ile Thr Leu Arg
Ala Glu Asp Asn Ala 85 90
95 Asp Thr Leu Ala Leu Val Phe Glu Ala Pro Asn Gln Glu Lys Val Ser
100 105 110 Asp Tyr
Glu Met Lys Leu Met Asp Leu Asp Val Glu Gln Leu Gly Ile 115
120 125 Pro Glu Gln Glu Tyr Ser Cys
Val Val Lys Met Pro Ser Gly Glu Phe 130 135
140 Ala Arg Ile Cys Arg Asp Leu Ser His Ile Gly Asp
Ala Val Val Ile 145 150 155
160 Ser Cys Ala Lys Asp Gly Val Lys Phe Ser Ala Ser Gly Glu Leu Gly
165 170 175 Asn Gly Asn
Ile Lys Leu Ser Gln Thr Ser Asn Val Asp Lys Glu Glu 180
185 190 Glu Ala Val Thr Ile Glu Met Asn
Glu Pro Val Gln Leu Thr Phe Ala 195 200
205 Leu Arg Tyr Leu Asn Phe Phe Thr Lys Ala Thr Pro Leu
Ser Ser Thr 210 215 220
Val Thr Leu Ser Met Ser Ala Asp Val Pro Leu Val Val Glu Tyr Lys 225
230 235 240 Ile Ala Asp Met
Gly His Leu Lys Tyr Tyr Leu Ala Pro Lys Ile Glu 245
250 255 Asp Glu Glu Gly Ser 260
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